The subject matter of this application provides novel recombinant telomerase enzyme genes and proteins and relates to the cloning and characterization of the catalytic protein component of mouse telomerase enzyme, referred to as mouse telomerase reverse transcriptase (xe2x80x9cmTERTxe2x80x9d).
This invention pertains generally to cell proliferation and aging, including the fields of age-related diseases, such as cancer and cell biology. In particular, this invention pertains to the discovery of a novel mTERT enzyme proteins and nucleic acids, and methods for isolating and expressing by recombinant means these nucleic acids and proteins. The invention provides antibodies specifically reactive with mTERT. The invention also pertains to methods of screening for novel mTERT activity modulators. The invention also includes means of mortalizing cells, creating indefinitely proliferating cells and immortalizing cells, including normal, diploid cells, using the novel reagents, proteins, nucleic acids, enzymes and methods of the invention.
The following discussion is intended to provide general information regarding the field of the present invention. The citation of various references is not to be construed as an admission of prior invention.
Telomeres, the protein-DNA structures physically located on the ends of chromosomes in eukaryotic organisms, are required for chromosome stability and are involved in chromosomal organization within the nucleus (Zakian (1995) Science 270:1601, Blackburn (1978) J. Mol. Biol., 120:33, Oka (1980) Gene 10:301, Klobutcher (1981) Proc. Natl. Acad. Sci. USA 78:3015). Telomeres are believed to be essential in most eukaryotes, as they allow cells to distinguish intact from broken chromosomes, protect chromosomes from degradation, and act as substrates for replication. Telomere loss, i.e., inability to maintain telomere structure, is associated with normal human cellular development, including cell aging and cellular senescence. Telomere gain, i.e., the ability to maintain telomere structure in cells, is associated with chromosomal changes and cancer.
Telomeres are generally replicated in a complex, cell cycle and developmentally regulated manner by a xe2x80x9cribonucleoprotein telomerase enzyme complex.xe2x80x9d The telomerase reverse transcriptase enzyme is a telomere-specific RNA-dependent DNA polymerase comprising a telomerase reverse transcriptase (TERT) protein and an RNA component. Telomerase enzyme uses its RNA component to specify the addition of telomeric DNA repeat sequences to chromosomal ends (U.S. Pat. No. 5,583,016; Villeponteau (1996) Cell and Develop. Biol. 7:15-21). In addition to the template RNA component, other proteins have been found to be associated with TRT. For example, telomerase-associated proteins called p80 and p95 were found in Tetrahymena (Collins (1995) Cell 81:677). Homologs of the p80 protein have been found in humans, rats and mice. Neither enzymatic activity nor amino acid motifs typically associated with RNA-dependent DNA polymerases have been found to be associated with these proteins (Harrington (1997) Science 275:973-977). In contrast, mutational analysis and reconstitution in vitro have shown the TERT proteins contain the catalytic moieties of telomerase (Lingner (1997) Science 276:561-567; Weinrich (1997) Nature Genetics 17:498-502). Various structural proteins that interact with telomeric DNA that are distinct from the protein components of TRT have also been described. In mammals, most of the simple repeated telomeric DNA is packaged in closely spaced nucleosomes (Makarov (1993) Cell 73:775, Tommerup (1994) Mol. Cell. Biol. 14:5777). However, the telomeric repeats located at the very ends of the human chromosomes appear to be in a non-nucleosomal structure that has been termed the telosome.
Telomeric DNA can consist of a variety of different structures. Typically, telomeres are tandem arrays of very simple sequences, such as simple repetitive sequences rich in G residues, in the strand that runs 5xe2x80x2 to 3xe2x80x2 toward the chromosomal end. In humans, the telomere repeat sequence is 5xe2x80x2-TTAGGG-3xe2x80x2 (SEQ ID NO:7). In contrast, telomeric DNA in Tetrahymena is comprised of repeats of the sequence T2G4, while in Oxytricha, the repeat sequence is T4G4 (Zakian (1995) Science 270:1601; Lingner (1994) Genes Develop. 8:1984). Heterogenous telomeric sequences have been reported in some organisms, such as the repeat sequence TG1-3 in Saccharomyces. The repeated telomeric sequence in other organisms is much longer, such as the 25 base pair repeat sequence of Kluyveromyces lactis. Furthermore, telomeric structure can be completely different in other organisms. For example, the telomeres of Drosophila are comprised of a transposable element (Biessman (1990) Cell 61:663, Sheen (1994) Proc. Natl. Acad. Sci. USA 91:12510).
In most organisms, the size of the telomere fluctuates. For example, the amount of telomeric DNA at individual yeast telomeres in a wild-type strain may range from approximately 200 to 400 bp, with this amount of DNA increasing and decreasing stochastically (Shampay (1988) Proc. Natl. Acad. Sci. USA 85:534). Heterogeneity and spontaneous changes in telomere length may reflect a complex balance between the processes involved in degradation and lengthening of telomeric tracts. In addition, genetic, nutritional and other factors may cause increases or decreases in telomeric length (Lustig (1986) Proc. Natl. Acad. Sci. USA 83:1398, Sandell (1994) Cell 91:12061).
Telomeres are not maintained via conventional replicative processes. Complete replication of the ends of linear eukaryotic chromosomes presents special problems for conventional methods of DNA replication. Conventional DNA polymerases cannot begin DNA synthesis de novo; rather, they require RNA primers that are later removed during replication. In the case of telomeres, removal of the RNA primer from the lagging-strand end would necessarily leave a 5xe2x80x2-terminal gap, resulting in the loss of sequence from the leading strand if the daughter telomere was subsequently blunt-ended (Watson, (1972) Nature New Biol. 239:197, Olovnikov (1973) J. Theor. Biol., 41:181).
While conventional DNA polymerases cannot accurately reproduce chromosomal DNA ends, specialized factors exist to ensure their complete replication. The telomerase enzyme is a key component in this process. In vivo, telomerase enzyme is assembled as a ribonucleoprotein (RNP) enzyme complex. It is an RNA-dependent DNA polymerase that uses a portion of its internal RNA moiety as a template for telomere repeat DNA synthesis (Yu (1990) Nature 344:126; Singer (1994) Science 266:404; Autexier (1994) Genes Develop. 8:563; Gilley (1995) Genes Develop. 9:2214; McEachern (1995) Nature 367:403; Blackburn (1992) Ann. Rev. Biochem. 61:113; Greider (1996) Ann. Rev. Became. 65:337). A combination of factors, including telomerase processivity, frequency of action at individual telomeres, and the rate of degradation of telomeric DNA, contribute to the size of the telomeres (i.e., whether they are lengthened, shortened, or maintained at a certain size). In vitro, telomerases may be extremely processive; for example, Tetrahymena telomerase can add an average of approximately 500 bases to the G strand primer before dissociation of the enzyme (Greider (1991) Mol. Cell. Biol., 11:4572).
Telomere replication is regulated both by developmental and cell cycle factors. Telomere replication may play a signaling role in the cell cycle. For example, certain DNA structures or DNA-protein complex formations may act as a checkpoint to indicate that chromosomal replication has been completed (Wellinger (1993) Mol. Cell. Biol. 13:4057). Telomere length is also believed to serve as a mitotic clock, which serves to limit the replication potential of cells in vivo and in vitro.
In humans, telomerase activity is not detectable in most somatic tissues. Cell that express either no or only low amounts of telomerase, such as somatic cells, undergo progressive telomere shortening with increasing age (Harley (1990) Nature 345:458, Harley (1994) Cold Spring Harbor Symp. Quant. Biol. 59:307). Some non-transformed, non-immortal cells have detectable telomerase activity. Germline cells express telomerase as required to maintain telomeric structure of chromosomes passed from generation to generation (Greider, (1996) Annu. Rev. Became. 65:337). Low levels of telomerase activity have been detected in activated human B and T lymphocytes and hematopoietic progenitor cells (Keiko (1995) J. Immunol. 155:3711; Igarshi (1997) Blood 89:1299-1307; Igarashi (1996) Biochem. Biophys. Res. Commun. 219:649; Norrback (1996) Blood 88:222).
Immortalized cells, such as most cancer cells, express significantly higher levels of telomerase, allowing for stabilization of telomeric structure. Telomerase activity has been detected in about 85% of biopsies from more than 950 primary human tumors (Kim (1994) Science 266:2011; Hiyama (1995) Nature Med. 1:249-257; Counter (1992) EMBO J. 11:192). Telomerase activity has been detected in many cancers (Wellinger (1993) supra; Autexier (1996) Trends Biochem. Sci. 21:387). However, even in telomerase-positive cells, such as most cancer cells, the levels of telomerase are very low relative to housekeeping and structural proteins.
Because telomerase is expressed (albeit in low levels) in most human cancer cells and is negligibly expressed in other cell types, it is the only true pan-cancer cell marker identified to date. Thus, there exists a great need for inhibitors of telomerase activity, which would be ideal therapeutic compositions in the treatment of cancer or uncontrolled cell growth. Furthermore, loss of or inhibition of telomerase activity is associated with cellular senescence and may lead to cell death. Therefore, there exists a great need for methods and compositions capable of promoting or reconstituting telomerase activity which would be useful in treating age-related disease and anti-aging pharmaceuticals. The present invention fulfills these and other needs.
This invention has for the first time provided the identification, cloning and characterization of mouse telomerase reverse transcriptase (mTERT) proteins and nucleic acids. Mouse telomerase enzymes, including associated nucleic acids and other polypeptides, are further provided. Also, the invention provides novel reagents and methods complementing this significant achievement.
The invention provides for an isolated or recombinant nucleic acid encoding an mTERT, the protein defined as having a calculated molecular weight of between 50 and 150 kDa, and specifically binding to an antibody raised against the protein of SEQ ID NO:2, or a subsequence thereof, or having at least 60% amino acid sequence identity to an mTERT protein comprising SEQ ID NO:2. In one embodiment, the calculated molecular weight of the encoded mTERT protein is about 127 kDa. In further embodiments, the encoded protein has at least 80% amino acid sequence identity to a protein comprising SEQ ID NO:2, or, the encoded protein comprises SEQ ID NO:2.
In alternative embodiments, the invention provides for an isolated or recombinant nucleic acid which specifically hybridizes to SEQ ID NO:1 under stringent conditions, an isolated nucleic acid encoding a protein which specifically binds to an antibody directed against a protein comprising SEQ ID NO:2, and an isolated nucleic acid comprising either 10 to 15 or more nucleotides identical or exactly complementary to SEQ ID NO:1 or a nucleotide sequence encoding at least about five contiguous amino acids of an mTERT, wherein the TERT has an amino acid sequence as set forth in SEQ ID NO:2 or conservative substitutions of said amino acid sequence. In another embodiment, the invention provides an isolated nucleic acid encoding a fusion protein comprising an mTERT. The invention also provides a nucleic acid free of dideoxynucleotides, as well as nucleic acids comprising non-naturally occurring nucleotides. One embodiment provides for an isolated nucleic acid comprising a label and a nucleotide sequence of the invention.
The invention also provides for an isolated or recombinant peptide encoded by a recombinant or isolated nucleotide sequence encoding at least about five contiguous amino acids of an mTERT.
In another embodiment, the invention provides for an isolated or recombinant mTERT protein where the mTERT has a calculated molecular weight of about 50 to 150 kDa; and specifically binds to an antibody raised against a protein comprising SEQ ID NO:2, or subsequence thereof, or has 60% amino acid sequence identity to a protein comprising SEQ ID NO:2. The isolated or recombinant mTERT protein can have a calculated molecular weight of about 127 kDa, or the protein can comprise SEQ ID NO:2. In an alternative embodiments, the isolated or recombinant mTERT protein is encoded by a nucleic acid molecule which specifically hybridizes to SEQ ID NO:1; and, the isolated or recombinant mTERT protein, or subsequence thereof, can further comprise a fusion protein.
The invention provides for an isolated or recombinant antibody specifically immunoreactive under immunologically reactive conditions to an mTERT protein; the mTERT protein can comprise the sequence as set forth in SEQ ID NO:2. The invention also provides for an isolated or recombinant antibody, specifically immunoreactive under immunologically reactive conditions, to an mTERT protein encoded by the nucleic acid of claim 1; the nucleic acid can comprise the sequence as set forth in SEQ ID NO:1. The invention further provides for an isolated or recombinant mTERT protein which specifically binds to the anti-mTERT antibodies of the invention.
Alternative embodiments provide for a transfected cell comprising a heterologous gene encoding a mTERT protein or subsequence thereof; a transfected cell into which an exogenous nucleic acid sequence has been introduced, where the nucleic acid specifically hybridizes under stringent conditions to SEQ ID NO:1 or a nucleic acid of the invention as described herein, and the cell expresses the exogenous nucleic acid as an mTERT protein; and a transfected cell where the transfected cell is a karotypically normal diploid cell.
The invention also provides for an organism into which an exogenous nucleic acid sequence has been introduced, the nucleic acid specifically hybridizing under stringent conditions to a nucleic acid with a sequence as set forth in SEQ ID NO:1, or a nucleic acid of the invention as described herein, and the organism expresses the exogenous nucleic acid as a mouse TERT protein. The organism can express an exogenous nucleic acid comprising a nucleic acid of the invention. Alternatively, the organism expresses and translates an exogenous nucleic acid sequence into a mouse TERT protein, which can be expressed externally from the organism. The organism can be an insect, as a Spodoptera sp., Trichoplusia sp. or a Lymantria sp. The insect can specifically be a Spodoptera frugiperda, Trichoplusia ni or a Lymantria dispar. The organism can be a plant, a fungus or a yeast. If it is a yeast, the organism can be a Pichia sp., Hansenula sp., Torulopsis sp., Saccharomyces sp., or a Candida sp. The yeast can specifically be a Pichia pastoris, Hansenula polymorpha, Torulopsis holmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis, or a Candida pseudotropicalis. The organism can be a bacterium, such as Escherichia coli, Streptococcus cremoris, Streptococcus lactis, Streptococcus thermophilus, Leuconostoc citrovorum, Leuconostoc mesenteroides, Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacterium bifidum, Bifidobacteriu breve, or a Bifidobacterium longum. 
The invention also provides for an expression vector comprising a nucleic acid sequence which specifically hybridizes under stringent conditions to an mTERT encoding nucleic acid; the nucleic acid can have a sequence as set forth in SEQ ID NO:1.
The invention also provides for a transfected cell comprising a recombinant mTERT, wherein said cell is comprised in a transgenic non-human animal. The invention also provides for a transgenic animal which lacks a functional mTERT due to its being xe2x80x9cknocked outxe2x80x9d using recombinant methods and reagents of the invention. Such mTERT knockouts mice are especially useful in studying the effect of telomerase and in testing anti-cancer telomerase inhibitors, i.e., in mice comprising human tumor xenografts.
In one embodiment, the invention provides for a transgenic cell or non-human animal, and progeny thereof, wherein said animal comprises an endogenous mTERT gene which has been mutated by recombinant means with a nucleic acid comprising a subsequence of a nucleic acid encoding an mTERT or complementary to an mTERT. The transgenic cell or non-human animal can be deficient in at least one mTERT or telomerase enzyme activity, or completely lack all mTERT or telomerase enzyme activity. The transgenic cell or non-human animal can comprise an mTERT with a deficiency in activity which is a result of a mutated gene encoding an mTERT having a reduced level of a telomerase enzyme activity compared to a wild-type telomerase enzyme activity. The transgenic cell or non-human animal can contain a mutated mTERT gene comprising one or more mutations selected from the group consisting of a missense mutation, a substitution, a nonsense mutation, an insertion, or a deletion. The transgenic cell or non-human animal can be a mouse, i.e., of the family Muridae. In particular, M. spretus or M. musculus spp. are provided. The transgenic non-human animal can further comprise a human telomerase reverse transcriptase.
The invention further provides for a kit for the detection of a mouse TERT gene or polypeptide, the kit comprising a container containing a molecule which can be a TERT nucleic acid or subsequence thereof, a TERT polypeptide or subsequence thereof, or an anti-TERT antibody.
The invention also provides a method of determining whether a test compound is a modulator of mTERT or telomerase enzyme activity, the method comprising the steps of: providing a mouse TERT composition, contacting the TERT with the test compound and measuring the activity of the TERT, where a change in TERT activity in the presence of the test compound is an indicator of whether the test compound modulates mouse TERT or telomerase enzyme activity.
In a further embodiment, the method is carried out in a buffered aqueous solution comprising a template polynucleotide, an mTERT, a buffered aqueous solution compatible with telomerase enzyme activity, and sufficient additional nucleotide species necessary for telomerase-catalyzed polymerization of a DNA polynucleotide complementary to said template polynucleotide. This method can be carried out in a cell-free extract, an organism or a transgenic organism. In alternative embodiments of this method: the DNA is a telomere or comprises a telomeric sequence; the template polynucleotide is a mouse telomerase RNA (mTR, or mouse telomerase related component, or mTERC) or comprises an mTERC subsequence; the activity of the telomerase is measured by monitoring incorporation of a nucleotide label into DNA; the activity of the telomerase enzyme is measured by monitoring the change in rate of incorporation of nucleotides into the DNA; the activity of the telomerase enzyme can also be measured by monitoring the accumulation or loss of nucleotides into the DNA; the activity of the telomerase enzyme and mTERT can be further measured by monitoring the loss of the ability to bind to a telomerase-associated protein; the activity of the telomerase enzyme and mTERT is measured by monitoring the loss of the ability to bind to a nucleic acid; and, the activity of the mTERT is measured by monitoring the loss of the ability to bind to a chromosome.
The invention also includes a method where the test compound produces a statistically significant decrease in the activity of mTERT as compared to the relative amount of incorporated label in a parallel reaction lacking the agent, thereby determining that the agent is a telomerase enzyme or mTERT inhibitor or activator. The method can be used to determine if there is a change in telomerase enzyme or TERT activity using, e.g., a TRAP assay or using a quantitative polymerase chain reaction assay. The method can determine a change in telomerase enzyme and mTERT activity by measuring the accumulation or loss of telomere structure.
The invention provides for isolated and recombinant murine proteins and nucleic acids that include murine (mTERT) specific motifs (see FIGS. 4 and 5) and TERT specific xe2x80x9cmotifs.xe2x80x9d These motifs effect common telomerase structure and function and uniquely define members of the mTERT species of the invention. Novel reagents of the invention corresponding to these motif regions can be used in methods of the invention to generate unique murine peptides and nucleic acids, including complementary and antisense hybridization probes and primers, to identify additional mTERT, including mTERT isoforms, homologues and alleles.
Two mTERT proteins are considered to have a statistically significant sequence identity, i.e., having significant homology, at the amino acid level in a conserved region of the TERT protein, such as the motifs described above and in FIGS. 4, and 5, if, after adjusting for deletions, additions and the like, the conserved regions have about 20% to 30% sequence identity, as can be deduced or derived from FIGS. 4 or 5. However, this sequence identity can be higher, e.g., as high as about 40% to 50% or higher, if, e.g. the conserved region of comparison is shorter, i.e., a region of about 5 to about 10 consecutive amino acids. Furthermore, the skilled artisan can deduce or derive additional mTERT motifs, modifications of these mTERT motifs, and variations in the amount of sequence identity in a particular mTERT motif to determine whether a polypeptide or nucleic acid is a member of the mTERT species of the invention, and the like, by reference to the teachings and sequences of the invention, particularly including FIGS. 4 and 5.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.